365NEWSX
365NEWSX
Subscribe

Welcome

Ras drives malignancy through stem cell crosstalk with the microenvironment - Nature.com

Ras drives malignancy through stem cell crosstalk with the microenvironment - Nature.com

Ras drives malignancy through stem cell crosstalk with the microenvironment - Nature.com
Nov 30, 2022 33 mins, 51 secs

Squamous cell carcinomas are triggered by marked elevation of RAS–MAPK signalling and progression from benign papilloma to invasive malignancy1,2,3,4.

At tumour–stromal interfaces, a subset of tumour-initiating progenitors, the cancer stem cells, obtain increased resistance to chemotherapy and immunotherapy along this pathway5,6.

The distribution and changes in cancer stem cells during progression from a benign state to invasive squamous cell carcinoma remain unclear.

Here we show in mice that, after oncogenic RAS activation, cancer stem cells rewire their gene expression program and trigger self-propelling, aberrant signalling crosstalk with their tissue microenvironment that drives their malignant progression.

The non-genetic, dynamic cascade of intercellular exchanges involves downstream pathways that are often mutated in advanced metastatic squamous cell carcinomas with high mutational burden7.

Coupling our clonal skin HRASG12V mouse model with single-cell transcriptomics, chromatin landscaping, lentiviral reporters and lineage tracing, we show that aberrant crosstalk between cancer stem cells and their microenvironment triggers angiogenesis and TGFβ signalling, creating conditions that are conducive for hijacking leptin and leptin receptor signalling, which in turn launches downstream phosphoinositide 3-kinase (PI3K)–AKT–mTOR signalling during the benign-to-malignant transition.

By functionally examining each step in this pathway, we reveal how dynamic temporal crosstalk with the microenvironment orchestrated by the stem cells profoundly fuels this path to malignancy.

Squamous cell carcinomas (SCCs) are common life-threatening cancers of the stratified epithelia of skin, oral cavity, oesophagus and lungs1,8,9.

It is less clear whether and how in healthy tissues an oncogenic mutation in a stem cell can intrinsically stimulate environmental changes that may lessen the need for multi-step mutagenesis.

After performing deep single-cell RNA sequencing (scRNA-seq) to gain insights into the SCC cancer stem cell (CSC) signature, we trace its temporal origins and physiological importance.

We show that, after oncogenic RAS initiation, tissue stem cells begin an aberrant molecular dialogue with their surroundings, culminating in a considerable remodelling of the tumour microenvironment at the benign-to-malignant transition.

This provides fertile ground for stromal TGFβ-mediated induction of leptin receptor (Lepr) and vasculature-mediated elevation of tissue leptin, leading to LEPR–leptin signalling and PI3K–AKT–mTOR in CSCs to drive the invasive switch.

Triggered by oncogenic RAS, each step of this stem cell–microenvironment crosstalk cascade is essential for malignant progression, and it involves pathways that are often mutated in advanced SCCs with a high mutational burden.

Skin stem cells that acquire HRAS mutations go through a benign papilloma state before progressing to malignant, invasive SCCs20,21.

In tumours displaying a mixed phenotype, basal progenitors undergoing TGFβ signalling are enriched for CSCs with increased resistance to chemotherapy and immunotherapy, and the loss of TGFβ signalling reverts tumours to a benign state5,6,19.

As judged by immunofluorescence imaging and fluorescence-activated cell sorting (FACS), TGFβ signalling and the phosphorylation of its downstream target transcriptional cofactor SMAD2 (pSMAD2) were rare in papillomas but increased substantially in invasive SCC progenitors (Fig. 1b and Extended Data Fig. 1a,b).

Taken together with our previous analysis of mixed papilloma–SCC tumours6, this result provided an important temporal layer by linking TGFβ signalling to the progression of CSCs from benign to malignant states.

Postnatally, doxycycline activates rtTA3 and induces HRASG12V in these stem cells.

Tu, tumour; St, stroma.

b, Quantification of a collapsed z-stack of 3D whole-mount immunofluorescence images and FACS-purified mCherry+ITGA6high basal progenitors reveals increased TGFβ signalling as tumours progress to invasive SCCs (Extended Data Fig. 1b).

c, UMAP representations and unsupervised k-nearest-neighbour-based clustering of single-cell transcriptomes performed on pooled FACS-isolated integrinlow (spiked, 159 total suprabasal) and integrinhigh (bulk, 1,346 total basal) cells from invasive SCC tumours3

Clusters C2 and C3, basal progenitors; C1, suprabasal cells.

Note that mCherry (TGFβ reporter, dotted box) is enriched in, but not exclusive to, C2 (35.8% of all basal cell progenitors).

See also Extended Data Fig.

P values were calculated using DAVID bioinformatic analysis. See also Extended Data Fig.

Quantifications are of keratin 18+ cell abundance, proximity to vessels and distances with vessels.

Investigating deeper into the physiological relevance of this temporal change, we added a creERT2 transgene under the control of the SBE-driven reporter and, on the basis of tamoxifen-activated lineage-tracing, we found that, even though the TGFβ-reporter-positive cells were infrequent in papillomas, they contributed substantially to SCCs (Extended Data Fig. 1c).

To further dissect the differences, we performed scRNA-seq Smart-seq2 analysis of histologically prevalidated, uniformly invasive SCCs of which the progenitors had been enriched by FACS (Extended Data Fig. 1d and Supplementary Fig. 3).

Quality controls revealed sufficient transcriptome detection rates, with ~7,500 genes per cell and low mitochondrial gene contamination (Extended Data Fig. 1e,f).

Transcriptomes fell into three clusters: C1, Itga6lowItgb1lowCd44+ suprabasal cells that had been added as a reference and displayed SCC differentiation markers such as Krt6b; and C2 and C3 basal cells, both of which were Itga6highItgb1highCd44+ and were expressed at higher levels than normal skin stem cells (Fig. 1c and Extended Data Fig. 1g).

Thus, although enriched for TGFβ signalling, C2 cells were not defined solely by this marker.

However, this cluster also displayed many other transcripts that are not clearly aligned with previous SCC-CSC signatures (Fig. 1c and Extended Data Fig. 1g).

Of 1,894 transcripts enriched in basal SCC cells relative to differentiated tumour cells, 732 were specific to C2 (Supplementary Table 1).

To place our CSC signature in the context of tumour progression, we performed bulk RNA-seq analysis of FACS-purified basal progenitors from normal skin, papillomas and SCCs, each staged temporally and histologically before processing (Extended Data Fig. 2a,b and Supplementary Fig. 4).

Relative to their normal skin counterparts, pan-tumour basal cells upregulated 886 transcripts by at least twofold (adjusted P ≤ 0.05; Supplementary Table 2), whereas 562 transcripts were upregulated specifically during the transition from benign to malignant states (Supplementary Table 3).

Although a number of C2 transcripts were found in papillomas, many were induced in SCCs, as exemplified by Krt8 and Krt18 transcripts and substantiated by immunofluorescence analysis (Extended Data Fig. 2b,c).

Angiogenesis appeared at the top of this list, along with cell migration, wound healing, protein phosphorylation and intracellular signalling (Fig. 1d).

Uniform manifold approximation and projection (UMAP) plots highlighted the enrichment of angiogenesis genes in this cluster, many of which were upregulated during the benign–malignant transition (Extended Data Fig. 2d).

Consistent with the preponderance of secreted angiogenic factors, reconstructed 3D immunofluorescence images revealed a significant influx in CD31+ vasculature specifically at the benign-to-invasive SCC transition (Extended Data Fig. 3a,b).

Overall, whereas previous studies reported an increase in vasculature during the transition from normal skin stem cells to papillomas24, here we found a notable further increase in the vasculature specifically during the progression to SCCs.

Despite their temporal lineage relationship, TGFβ-responding basal SCC cells differed from those of papillomas (Extended Data Fig. 2e,f).

These data suggest that progenitors that progress to SCC are influenced by shifting crosstalk with their tumour microenvironment.

When C2 cells were specifically scored for elevated TGFβ signalling, 101 associated transcripts were also upregulated (Fig. 2a and Supplementary Table 4).

In addition to Cd80—a factor in resisting immunotherapy5—this shortlist included Ccnd1 and Ccnd2, Hmga2, Pcolce2, Rgs16, St8sia1, Tnfaip2 and Pthlh, which are known to correlate with stem cell self-renewal/survival, proliferation and/or poor prognosis in SCCs.

a, Venn diagram showing that 101 genes constitute a refined CSC signature shared by single-cell C2 and TGFβ-responsive transcriptomes in SCC basal progenitors (Extended Data Fig. 2).

b, Lepr-expressing cells reside within the C2 basal SCC population and overlap around 75% with TGFβ-reporter+ cells.

c, Immunofluorescence analysis of primary mouse skin SCC confirms that LEPR is rarely expressed in papillomas but is enriched in TGFβ-reporter+ SCC cells (arrowheads).

d, LEPR immunoblot analysis.

Cultured HrasG12V keratinocytes (KT) that are wild type (FF) but not mutant (ΔΔ) for the TGFβ receptor gene (Tgfbr2) elevate LEPR substantially in response to active recombinant TGFβ1.

e, Immunofluorescence analysis of tumour tissue from FR-LSL-HrasG12V;Tgfbr2fl/fl;R26-LSL-YFP mice transduced at a low titre with PGK-creERT2 lentivirus, and treated with tamoxifen to induce YFP(pseudoRed)+ HrasG12VTgfbr2ΔΔ tumorigenesis.

The loss of TGFβ signalling results in non-invasive tumours that do not express LEPR.

f, ATAC-seq was performed on FACS-purified ITGA6highITGB1high basal populations of interfollicular epidermis (IFE, SCA1+), bulge hair follicle stem cells (HFSCs, CD34+) and tumour cells (CD44high) either positive or negative for TGFβ responsiveness (mCherry).

ATAC peaks associated with the Lepr locus opened during tumorigenesis, with the encased cluster 6 peak (containing RUNX, AP1 and SMAD motifs) opening predominantly during SCC progression.

See also Extended Data Figs.

g, Schematic of the in vivo Lepr ATAC-peak eGFP reporter assay.

As TGFβ-signalling papilloma progenitors lineage traced to SCC-CSCs, these data implied that CSC gene expression is affected by changes in the tumour microenvironment.

In considering CSC signature proteins that might be able to sense, respond to and take advantage of the notable changes in the tumour microenvironment at the benign–malignant transition, Lepr caught our attention.

Traditionally studied in the context of energy balance, LEPR signalling is triggered by its ligand leptin, which is primarily produced by white adipose tissue, but can enter the circulation to reach distal LEPR+ target tissues, such as the hypothalamus25.

Within SCC progenitors, Lepr was specifically transcribed in mCherry+ TGFβ-signalling C2 CSCs (Fig. 2a,b and Extended Data Fig. 2g).

LEPR immunofluorescence corroborated its location in invasive mouse SCCs, and was also found human SCC tumours and xenografts (Fig. 2c and Extended Data Fig. 4a).

To understand the specificity of Lepr to C2 TGFβ-signalling cells, we first exposed cultured isogenic TGFβ receptor floxed and null HrasG12V keratinocytes to recombinant TGFβ1 or vehicle control.

Immunoblot analysis underscored the sensitivity of LEPR to TGFβ signalling (Fig. 2d).

Without TGFβ receptor signalling, which is known to be essential for EMT-mediated invasion6, only a few rare LEPR+ cells were detected by immunofluorescence (Fig. 2e).

To address whether Lepr is a direct transcriptional target of TGFβ receptor signalling in vivo, we performed an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) analysis of FACS-purified TGFβ-reporter positive versus negative basal tumour populations (Extended Data Fig. 4b–d).

Unsupervised clustering of ATAC profiles from purified progenitors of normal skin (interfollicular epidermis; hair follicle), papilloma and SCC revealed seven clusters (Extended Data Fig. 5a).

Peaks in the proximity of Lepr mostly fell into clusters 4 and 6, of which the chromatin state displayed marked opening during tumorigenesis, particularly in association with TGFβ-signalling CSCs (Fig. 2f and Extended Data Fig. 5b,c).

Notably, Lepr was among the genes bearing such ATAC peaks and of which the accessibility was sensitive to TGFβ signalling and malignant progression (Fig. 2f and Extended Data Fig. 5d).

Notably, RUNX1 has been shown to be critical for tumour initiation26, whereas elevated AP1 (FOS) has been shown to drive basal cell carcinoma to more aggressive SCCs27.

Similar to pSMAD2, both RUNX1 and FOS showed marked nuclear localization in SCC basal cells at invasive fronts (Extended Data Figs. 1a,b and 5e).

To directly test whether tumour-stage-specific changes in TGFβ signalling govern the chromatin accessibility and expression of Lepr, we examined the ability of the C6 cis-regulatory element (Fig. 2f, magenta box) to drive temporal activation of an eGFP reporter during tumorigenesis.

Interestingly, the Lepr reporter was highly active at invasive SCC fronts where TGFβ signalling is high6, while much lower in papillomas (Fig. 2g).

Consistent with this correlation, in utero co-injection of a TGFβ-signalling mCherrynuclear reporter and a Lepr-eGFPcytoplasmic reporter revealed that the highest double-fluorescence positivity was among invasive SCC, and the majority of total TGFβ-signalling cells in these regions were positive for the Lepr-eGFPcytoplasmic reporter in SCC in contrast to papilloma (Extended Data Fig. 5f).

These data further underscore the physiological relevance of TGFβ signalling in fuelling the epigenetic dynamics that lead to Lepr promoter activation during the transition from the benign to malignant states.

Given the association between Lepr and C2 SCC-CSCs, we next performed colony-forming assays to test for stemness and found that LEPR+ C2 cells showed nearly a threefold higher colony-forming efficiency and formed larger colonies compared with LEPR− C3 cells (Fig. 3a).

To functionally test LEPR’s tumour-initiating ability in vivo, we turned to a highly aggressive mouse SCC cell line containing mutations in Hras and Trp5328 (hereafter referred to as PDV).

After verifying the TGFβ sensitivity with the Lepr reporter in these cells, we used CRISPR–Cas9 editing to generate a Lepr-null mutation (Extended Data Fig. 6a,b).

Serial-dilution orthotopic transplantation assays on Leprnull and Leprctrl PDV cells intradermally injected into immunocompromised Nude mice revealed an approximately 10× higher tumour-initiating ability if LEPR was intact (Fig. 3b).

Overall, these results suggested that LEPR identifies a subpopulation of TGFβ-signalling, oncogenic-RAS-driven SCC progenitors endowed with heightened stemness and tumour-initiating ability.

a, Stem cell colony assay.

Leprnull PDVC57 (PDV) SCC cells were generated by CRISPR–Cas9 gene editing (Extended Data Fig. 6b).

c, Leptin receptor deficiency impairs SCC progression.

Leprnull PDV tumours display reduced growth compared with their control counterparts (n = 4, P = 0.0039 for the end timepoint).

d, LEPR signalling functions in SCC progression.

Lentiviruses containing doxycycline (doxy)-inducible versions of either full-length (FL) Lepr or LeprΔsig were transduced into Leprnull PDV SCC cells expressing rtTA3 (required for doxycycline-induced activation of the TRE) (Extended Data Fig. 6d).

Leprnull PDV tumour growth is robust only when full-length Lepr but not LeprΔsig is reintroduced into tumour cells (n = 6, P = 0.0008 for the end timepoint), underscoring the need for active LEPR signalling, and not merely LEPR, in tumour growth.

As judged by labelling of S-phase cells with thymidine analogue 5-ethynyl-2′-deoxyuridine (EdU), Lepr loss reduced, although did not abrogate, proliferation within the tumour (Extended Data Fig. 6c).

To test whether active LEPR-signalling is required to drive SCC progression, we asked whether we could rescue the inhibitory effects of Lepr ablation with an inducible Lepr transgene that lacked the encoded cytoplasmic signalling domain of LEPR (ΔSig).

Allografts on non-obese host mice revealed that even though transduced full-length LEPR was expressed at lower levels than the control, it restored aggressive SCC tumour growth to PDV Leprnull cells.

By contrast, the expression of LEPR(ΔSig) had little if any effect (Fig. 3d and Extended Data Fig. 6d).

Thus, LEPR signalling, and not merely the presence of LEPR, was critical in driving SCC progression of RAS-driven oncogenic stem cells.

As judged by tumour lysate ELISAs, leptin levels were greater than 5× higher in total tumour tissue of SCC relative to papilloma (Fig. 4a).

This rise emanated from the tumour microenvironment, as neither the epithelial papilloma nor SCC cells expressed the ligand (Extended Data Fig. 2g).

Turning to the source of elevated leptin, we first considered direct delivery from local fat, but saw no overt signs of increased adipogenesis in the tumour microenvironment as judged by Oil red O staining (Fig. 4b).

Leptin in tumour tissue lysates is elevated as papillomas progress to SCC.

c, Quantitative PCR reveals no significant Lep transcriptional differences in the tumour microenvironments of SCCs versus papillomas.

e, Tumour growth and angiogenesis are enhanced by intradermal recombinant mouse VEGFA (rmVEGFA), injected every 3 days into PDV SCC tumours and assayed beginning at day 21 after grafting.

VEGFA increases the CD31+ tumour vasculature, as judged by flow cytometry.

f, Elevated expression of SCC stem cell C2 signature gene Vegfa is sufficient to enhance local angiogenesis and elevate leptin levels in the tumour microenvironment.

Scale bars, 50 µm. g, SCC tumour growth is sensitive to plasma leptin levels.

Recombinant leptin or mutant SMLA leptin agonist (doses indicated) was delivered to the circulation by an osmotic pump and the effects on PDV SCC tumour growth were monitored for 5 weeks.

We first used an osmotic pump to deliver fluorescently labelled leptin to the circulation and verified leptin’s ability to enter both normal skin dermis and tumour stroma from circulation (Extended Data Fig. 7a).

However, in contrast to obese animals, in which serum leptin is elevated25,29, our tumour-bearing mice were not obese, and we did not detect a significant increase in serum leptin during tumour progression (Fig. 4d).

To test this possibility, we first intradermally injected recombinant VEGFA and verified that both angiogenesis and tumour growth were markedly increased (Fig. 4e).

To guard against wound-induced effects due to injections, we also validated these effects by osmotic pump implantation to deliver VEGFA systemically (Extended Data Fig. 7b).

As increasing capillary density might elevate additional hormones and growth factors within the tissue, we used an osmotic pump to directly manipulate leptin levels in the circulation.

Using different doses of recombinant leptin as well as a superactive mouse leptin antagonist (SMLA) that abrogates leptin’s signalling activity even when bound to LEPR30, we further found that circulating leptin accelerated tumour growth in a dose-dependent manner, whereas SMLA had a slightly repressive effect (Fig. 4g).

These findings underscored the ability of circulating leptin on its own to affect tumour progression.

That said, there was a measurable modest difference, raising the possibility that a feed-forward loop may be operating during malignant progression (Extended Data Fig. 7c).

Overall, when coupled with the enhanced proximity of Lepr reporter activity to blood vessels in SCC-CSCs (Extended Data Fig. 7d), our results provide compelling support for a model in which increased angiogenesis at the invasive SCC front endows the tumour microenvironment with an ample supply of leptin, while perivascular-associated immune and other stromal cells6,19 provide the TGFβ necessary to induce Lepr expression in CSCs.

In other cellular contexts, LEPR signalling relies on its association with the Janus kinase (JAK2), which, after leptin-LEPR binding, phosphorylates LEPR’s intracellular domain.

Note the leptin-dependent activation of pAKT exclusively in LEPR+ cells, along with higher AKT levels (Extended Data Fig. 8e).

d, Immunocompromised mice with Leprctrl and Leprnull PDV tumours on opposite sides of their backs were administered the PI3K inhibitor BKM120 or vehicle control daily through oral gavage beginning at 14 days after PDVC57 cell injections.

As judged by this assay, most tumour growth attributable to PI3K signalling operates through LEPR.

f, The importance of leptin–LEPR signalling in activating mTORC1 signalling is accentuated in vivo, where the background from other growth factors in enriched medium is eliminated.

pS6 immunofluorescence reveals LEPR dependency on mTORC1 activity in PDV-engrafted tumours and particularly pronounced activity at the invading fronts of LEPR+ HRASG12V SCCs.

g, pS6 immunofluorescence (mTORC1 activity) and Lepr eGFP reporter (rep) activity co-localize in cells at invading HRASG12V SCC fronts.

h, Immunocompromised mice with Leprctrl and Leprnull PDV tumours on opposite sides of their backs were continuously administered rapamycin or vehicle control at t = 3 weeks and then monitored for tumour progression.

As judged by this assay, most tumour growth attributable to mTOR signalling operates through LEPR.

At the transcriptional level, the JAK–STAT signature showed no enrichment in our SCC-CSCs and, although flow cytometry verified JAK2 phosphorylation, the differences between papilloma and SCC, while variable, were not significant (Extended Data Fig. 8a,b).

STAT3 was also phosphorylated and present in the nucleus in papillomas and, although pSTAT3 was diminished in Leprnull PDV tumours, it was not abrogated (Extended Data Fig. 8c).

Thus, LEPR–leptin signalling appeared to act as a catalyst to enhance, not induce, JAK–STAT signalling to a level that facilitated progression from the benign to invasive state in SCCs.

Turning to an unbiased approach to delve further into mechanism, we analysed our transcriptomes of individual SCC basal cells according to their level of Lepr expression.

On the basis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the top three pathways distinguishing Lepr+ versus Lepr− basal SCC cells were small-cell lung cancer (oncogenic RAS-associated), pathways in cancer and the PI3K–AKT signalling pathway (Fig. 5b, top).

Indeed, comprehensive gene signature expression scores for the AKT signalling pathway showed significant upregulation in C2 SCC-CSCs, with a marked elevation between papilloma and SCC states (Extended Data Fig. 8d,e).

To examine the PI3K–AKT connection further, we performed bulk RNA-seq analysis of FACS-purified basal cells from tumours that developed from our engrafted PDVC57 cells.

Taken together, LEPR-PI3K–AKT surfaced as a top candidate for a signalling pathway that could account for the heterogeneity in our basal progenitor population of invasive SCCs.

In vitro, Leprctrl but not Leprnull SCC cells were sensitive to AKT–PI3K signalling in the presence of recombinant leptin.

As judged by immunoblot analyses, both AKT stability and activation (phosphorylation) were enhanced by leptin, but only if SCC cells expressed LEPR (Fig. 5c).

Moreover, when we blocked PI3K signalling directly in vivo, the oral PI3K inhibitor BKM12033 reduced tumour growth in only Leprctrl SCC and not in Leprnull SCC (Fig. 5d).

Considering the many routes through which PI3K–AKT can be activated, its robust link to LEPR signalling in driving oncogenic RAS tumours to an invasive SCC state was surprising and suggested that, in this context, LEPR–leptin signalling has a profound role in orchestrating the PI3K–AKT cascade and fuelling SCC tumour growth.

In agreement, Leprctrl PDV cells in vitro were larger in size compared with Leprnull PDV cells (Extended Data Fig. 8f).

The importance of LEPR in regulating PI3K–AKT–mTOR in SCC-CSCs extended to in vivo tumours.

Furthermore, in the HRAS(G12V)-mediated transition from papilloma to SCC, pS6 was elevated at invasive SCC fronts and, when imaged with our Lepr reporter, pS6 and eGFP showed considerable overlap in these regions (Fig. 5g).

Finally, continuous delivery of the potent mTOR inhibitor rapamycin resulted in reduced growth of tumours derived from engrafted Leprctrl PDV cells (Fig. 5h).

By contrast, rapamycin had less effect on Leprnull tumours, the growth of which was already restricted by LEPR loss of function.

Notably, although the GO terms for LEPR sensitivity pointed to the PI3K–AKT pathway, AKT can also be phosphorylated by mTORC1, leaving open the possibility of feedback mechanisms arising downstream of LEPR signalling.

However, the tumour microenvironment can be equally impactful in driving malignant progression, as exemplified by the effects of obesity on cancer14,38,39.

In the attempt to identify obesity-driven tumour susceptibility pathways that might alter energy balance, leptin–LEPR signalling has been a focus of cancers of which the normal stem cells express LEPR and exist in a fatty tissue microenvironment in which local leptin is high35,40,41.

For cancers such as SCCs that originate from native tissues that do not express LEPR, reports of LEPR expression have relied mostly on immunolabelling with antibodies of unclear specificity42,43,44.

How alterations in LEPR signalling contribute to tumour progression and metastasis has remained unclear.

Mechanistic insights have relied on cultured cancer cell lines, in which different possible routes have been proposed35,43,45 (Fig. 5a).

Moreover, it was recently demonstrated that obesity generated by leptin deficiency in mice can affect KRAS-induced pancreatic cancer progression not through impaired LEPR-signalling but, rather, through an obesity-specific mechanism involving aberrant endocrine–exocrine signalling in the adapting pancreatic beta cells14.

Rather, we uncovered a cancer link to the leptin–LEPR signalling pathway that becomes activated de novo downstream of an oncogenic HRAS(G12V)-induced change within otherwise normal skin stem cells.

In marked contrast to oncogenic KRAS-induced pancreatic cancers, which are influenced heavily by obesity but not leptin14, or to pathogen infections that can elicit transient changes in local adipose tissue/leptin levels that affect wound repair46, malignant progression in HRAS-induced cutaneous cancers requires the induction of LEPR signalling by the stem cells, but neither obesity nor adipogenesis in the local tissue environment.

LEPR signalling during SCC progression appears to be rooted in two events: first, a CSC-mediated influx of vasculature within the tumour microenvironment that increases blood vessel density at the invasive front and in turn causes local leptin levels to rise within the tumour stroma; and second, a corresponding rise in perivascular TGFβ that enhances TGFβ signalling and Lepr gene expression within neighbouring SCC-CSCs.

Thus, through the ability of oncogenic RAS to reroute the stem cell’s communication circuitry with its surrounding microenvironment, and the ability of the microenvironment in turn to induce a membrane receptor on the stem cells, CSCs exploit this dynamic crosstalk, fuelling non-genetic circuitries that drive malignant progression (Extended Data Fig. 8g).

In summary, the acquisition of an oncogenic RAS mutation sparks the perfect crosstalk between tumour-initiating cells and their microenvironment, enabling them to hijack the LEPR-signalling pathway and fuel cancer progression.

In this regard, the downstream consequences of LEPR signalling, namely sustained activation of the PI3K–AKT–mTOR pathway, become all the more important because, among human cancers, PI3KCA is among the most commonly mutated genes and a target of emerging anti-cancer therapeutics7,36.

Similarly, although polymorphisms in Lep and Lepr have been associated with oral SCCs47, our data clearly show that, even if a causal link emerges in the future, such genetic alterations are not required to initiate signalling.

The mouse cutaneous SCC cell line PDVC57 was cultured in the E-low medium (E.F.’s laboratory)5.

Mouse keratinocyte cell line FF (Tgfbr2f/fPGK-HrasG12V) and ΔΔ (Tgfbr2nullPGK-HrasG12V) were cultured with the E-low medium as previously discribed6.

The HNSCC cell line A431 was cultured in DMEM medium (Gibco) with 10% FCS, 100 U ml−1 streptomycin and 100 mg ml−1 penicillin.

The HEK 293TN cell line for lentiviral production was cultured in DMEM medium supplemented with 10% FCS (Gibco), 1 mM sodium pyruvate, 2 mM glutamine, 100 U ml−1 streptomycin and 100 mg ml−1 penicillin.

The 3T3J2 fibroblast feeder cell line was expanded in DMEM/F12 medium (Thermo Fisher Scientific) with 10% CFS (Gibco), 100 U ml−1 streptomycin and 100 mg ml−1 penicillin.

It was then treated with 10 µg ml−1 mitomycin C (Sigma-Aldrich) for 2 h to achieve growth inhibition.

The human skin SCC line A431 was from ATCC; mouse skin SCC PDVC57 was a gift from the original laboratory that created it (Balmain lab); mouse keratinocyte cell lines FF (Tgfbr2f/fPGK-HrasG12V) and ΔΔ (Tgfbr2null PGK-HrasG12V) were generated in E.F.’s laboratory; mouse fibroblast 3T3/J2 has been passaged in the laboratory as feeder cells and originated from the laboratory of H.

PDVC57 was validated by karyotyping and grafting tests.

Mouse keratinocyte cell lines were validated previously in E.F.’s laboratory.

Lentiviral constructs were previously described (SBE-NLSmCherry-P2A-CreERT2 PGK-rtTA3)6 or cloned in E.F.’s laboratory (SBE-NLSmCherry PGK-rtTA3, Lepr peak reporter-eGFP PGK-rtTA3, TRE-Lepr-IRES-eGFP, PGK-rtTA3, TRE-Vegfa EEF1A1-rtTA3, TRE-STOP EEF1A1-rtTA3).

For tumour allograft studies, 1 × 105 mouse PDVC57 SCC cells were mixed with growth-factor-reduced Matrigel (Corning) and intradermally injected into NU/NU Nude immunocompromised mice.

For metastatic tumour xenografts, 1 × 105 human SCC A431 cells were resuspended in sterile PBS and tail-vein injected into immunocompromised Nude mice.

After blocking, the sections were stained with primary antibodies: ITGA6 (rat, 1:2,000, BD), RFP/mCherry (guinea pig, 1:5,000, E.F.’s laboratory), K14 (chicken, 1:1,000, BioLegend), CD31 (rat, 1:100, BD Biosciences), K5 (guinea pig, 1:2,000, E.F.’s laboratory), K8 (rabbit, 1:1,000, E.F.’s laboratory), mLEPR (goat, 1:200, R&D Systems), hLEPR (rabbit, 1:100, Sigma-Aldrich), RUNX1 (rabbit, 1:100, Abcam), FOS (rabbit, 1:100, Cell Signalling), GFP (chicken, 1:500, BioLegend), pSTAT3-Y705 (rabbit, 1:100, Cell Signalling), pSMAS2-S465/467 (rabbit, 1:1,000, Cell Signalling) or pS6-S240/244 (rabbit, 1:100, Cell Signalling).

The shortest distance and volume measurements were performed by the creation of individual objects of CD31+ blood vessels, K14+ tumour mass, K18+ tumour cells or Lepr reporter+ tumour cells.

To sort the target tumour cell populations by FACS, tumours were first dissected from the skin and finely minced in 0.25% collagenase (Sigma-Aldrich) in HBSS (Gibco) solution.

The pellet was resuspended in 20 ml FACS buffer and strained through a 70 μm cell strainer (BD Biosciences).

The cell pellet was then resuspended in primary antibodies.

To sort the skin stem cell populations (IFE and HFSCs), whole back skins were first dissected from the mouse.

For the in vivo Lepr reporter SCC cell experiment, reporter PDVC57 cells were treated with TGFβ1 (10 ng ml−1) for 7 days.

The treated reporter PDVC57 cells were stained with 100 ng ml−1 DAPI in FACS buffer and analysed on the BD Biosciences LSR Fortessa system together with the control treatment (BSA only).

For bulk RNA-seq, targeted cell populations from 2 (SCC) to 15 (papilloma) tumours per population were directly sorted into TRI Reagent (Thermo Fisher Scientific) and the total RNA was purified using the Direct-zol RNA MiniPrep Kit (Zymo Research) according to the manufacturer’s instructions.

For accessible chromatin profiling, target cell populations from 2 (SCC) to 15 (papilloma) tumours per population were sorted into FACS buffer, and ATAC-seq sample preparation was performed as described previously51.

For scRNA-seq, target cell populations were sorted from 3–5 SCC tumours per mouse, for a total of 3 biological replicates (2 male and 1 female mice).

Sequencing reads per cell from each lane were combined during alignment to the reference genome.

Our Leprnull PDVC57 cell line was generated using the Alt-R CRISPR–Cas9 system (IDT).

In brief, a recombinant Cas9 protein, validated sgRNA (GAGUCAUCGGUUGUGUUCGG) targeting exon 3 of the mouse Lepr gene or a negative control sgRNA (IDT), and an ATTO-550-conjugated tracer RNA were used to form a ribonucleoprotein (RNP) were mixed with RNAiMax reagent (Thermo Fisher Scientific).

PDVC57 cells were then transfected with the mixture overnight and FACS-purified into 96-well plates to produce clonal cell lines.

The Leprnull PDVC57 cell line was selected after validating by immunoblot analysis of LEPR as well as sequencing of the target region for indel efficiency using the MiSeq system.

The Leprnull PDVC57 cell line and Leprctrl PDVC57 cell line were intradermally injected into the immunocompromised Nude mice, and the tumours were analysed for growth and progression.

Leprnull PDVC57 cells were transduced in vitro with 1:1 ratio of PGK-rtTA3 lentivirus and TRE-FL-Lepr-IRES-eGFP or TRE-LeprΔSig-IRES-eGFP lentivirus.

After culturing in 1 μg ml−1 of doxycycline (Sigma-Aldrich) containing E-Low medium, eGFPhigh cells expressing Lepr were isolated by FACS and expanded in vitro.

These two different cell lines were later intradermally grafted onto immunocompromised Nude mice, and the tumours were analysed for growth and progression.

To compare the tumour-initiating ability between Leprnull PDVC57 and Leprctrl PDVC57 cell lines, a preset number of cells were intradermally grafted onto Nude mice, and the tumour growth was tracked for 5 weeks to calculate the tumorigenicity of cells.

As previously described23, SCC cells were diluted serially from 104 to 106 cells per ml and 100 µl cell mixtures in 1:1 PBS:Matrigel were injected.

Tumour sizes were then monitored for tumour growth and progression.

To measure Lep levels in specific cells from the tumour or normal microenvironment, CD45+ (immune cells), CD140a+ (fibroblasts and other mesenchymal cells), CD117+ (melanocytes) and CD31+ (endothelial cells) were FACS-isolated from single-cell suspensions of normal skin, papilloma and SCC in Tri-Reagent (Thermo Fisher Scientific).

After LPER+ and LEPR− tumour basal cells (CD29/CD49fhighCD44+) were FACS isolated and counted, 5 × 104 cells of each replicate per condition were plated in a 10 cm dish with a growth-inhibited 3T3/J2 feeder layer with the SY medium (E.F.’s laboratory, see below) at 7.5% CO2 and 37 °C.

The following primary antibodies and dilutions were used: primary antibodies (anti-mLEPR 1:1,000, R&D Systems; anti-AKT, 1:1,000, Cell Signaling; anti-pAKT(S473), 1:1,000, Cell Signalling; anti-S6, 1:1,000, Cell Signaling; anti-pS6(S240/244), 1:1,000, Cell Signaling; anti-S6K, 1:1,000, R&D Systems; anti-pS6K(T389), 1:1,000, Cell Signaling; anti-GAPDH, 1:5,000, Thermo Fisher Scientific; anti-α-tubulin, 1:5,000, Sigma-Aldrich), secondary antibodies were used at 1:10,000 (donkey anti-rabbit HRP and donkey anti-mouse Alexa647, Jackson ImmunoResearch).

For RNA-seq analysis of C57BL/6J TRE-HRASG12V driven papilloma and SCC samples (Fig. 1 and Extended Data Fig. 1), raw sequencing reads were aligned to the mouse reference genome (UCSC release mm10) using Bowtie2 (v.2.2.9)55 using the default parameters.

For downstream analyses, cells with <2,500 genes detected per cell and genes expressed in <5% of the cell population were removed.

After filtering, there were 1,504 cells (159 integrinlow suprabasal, 500 integrinhigh, mCherry− basal, and 845 integrinhigh, mCherry+ basal cells) (n = 3 mice) in the dataset.

To identify cell clusters and visualize the data, we first centred and scaled the highly variable gene dataset and performed principal component analysis on the list of highly variable genes.

The resulting gene set scores for each cell were colour coded on corresponding UMAP visualizations of the data.

All of the error bars in the box plots and growth curves are mean ± s.e.m.

Squamous cell cancers: a unified perspective on biology and genetics.

Cancer Cell 29, 622–637 (2016).

Analysis of the rasH oncogene and its p21 product in chemically induced skin tumors and tumor-derived cell lines.

Genomic landscape of carcinogen-induced and genetically induced mouse skin squamous cell carcinoma.

Adaptive immune resistance emerges from tumor-initiating stem cells.

Cell 177, 1172–1186 (2019).

TGF-beta promotes heterogeneity and drug resistance in squamous cell carcinoma.

Cell 160, 963–976 (2015).

Squamous cell carcinoma—similarities and differences among anatomical sites.

Age-induced accumulation of methylmalonic acid promotes tumour progression.

Skin squamous cell carcinoma propagating cells increase with tumour progression and invasiveness.

Tumor-initiating stem cells of squamous cell carcinomas and their control by TGF-beta and integrin/focal adhesion kinase (FAK) signaling.

Defining a tissue stem cell-driven Runx1/Stat3 signalling axis in epithelial cancerJ

c-FOS drives reversible basal to squamous cell carcinoma transition.

Cell Rep.

Alterations in signal transduction pathways implicated in tumour progression during multistage mouse skin carcinogenesis.

CellM

Cytokine Growth Factor Rev.

Ras, PI(3)K and mTOR signalling controls tumour cell growth.

Leptin—a growth factor in normal and malignant breast cells and for normal mammary gland development.

Disruption of leptin receptor expression in the pancreas directly affects beta cell growth and function in mice.

Gallic acid modulates phenotypic behavior and gene expression in oral squamous cell carcinoma cells by interfering with leptin pathway.

Leptin acts on neoplastic behavior and expression levels of genes related to hypoxia, angiogenesis, and invasiveness in oral squamous cell carcinoma.

Clinical–pathological significance of leptin receptor (LEPR) expression in squamous cell carcinoma of the skin.

Leptin impairs the therapeutic effect of ionizing radiation in oral squamous cell carcinoma cells.

A study on oncogenic role of leptin and leptin receptor in oral squamous cell.

RNAi screens in mice identify physiological regulators of oncogenic growth.

RSEM: accurate transcript quantification from RNA-seq data with or without a reference genome.

Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities.

Cell 38, 576–589 (2010).

Huang for scRNA-seq and raw data analyses; and A

Balmain for PDVC57 cells

is the recipient of a New York Stem Cell Foundation Druckenmiller postdoctoral fellowship; K.T

The FACS instruments used in the study were supported by the Empire State Stem Cell fund through NYSDOH (C023046)

Robin Chemers Neustein Laboratory of Mammalian Cell Biology and Development, The Rockefeller University, New York, NY, USA

designed the experiments and interpreted the data

participated in all mouse tumour cell engraftments

performed PDVC57 and Tgfbr2 cKO-related experiments

analysed the scRNA-seq data

orchestrated all of the in vivo tumorigenesis experiments, performed in vitro cell cultures, and prepared samples for the sequencing, ELISA and immunofluorescence analysis

b, Immunofluorescence of sagittal tumour sections for TGFβ reporter (mCherry) signalling, α6 integrin to demarcate the tumour-stromal border and DAPI (nuclei)

c, Lineage tracing of TGFβ-signalling tumour cells marked at the papilloma stage, traced to the SCC and analysed by FACS shows that TGFβ-responding papilloma cells contribute significantly to SCC tumour progression

e, UMAP of the number of genes per cell

f, Violin plots showing that the quality of samples (with FACS labelled cell identities) in the scRNAseq was high as judged by the number of counts per cell, the number of genes per cell, and the low percentage of the mitochondrial genome captured

g, UMAPs of control genes for basal SCC cells (Itga6, Cd44), suprabasal tumour cells (Krt6b) and SCC-CSCs (Cd80) (see Fig. 1c for additional details). All statistics were using unpaired two-tailed Student’s t-test: ns, p ≥ 0.05); *, p ≤ 0.05); **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001

Data are presented as mean ± s.e.m. The diagram in c was created using BioRender

Source data

Basal cells (n = 2 mice per condition) are isolated by FACS using tumour basal cell markers (ITGA6, ITGB1, and CD44) with non-epithelial cell types (CD31, endothelial cells; CD45 pan-immune cells; CD117, melanocytes; CD140a, mesenchymal cells) excluded

b, Heatmap representation of bulk RNAseq of FACS-isolated basal progenitors from normal skin epithelia, papilloma, and SCC (in replicate) show significant molecular changes and stage-specific signatures during tumour progression

c, Immunofluorescence images show that keratin 8 positive tumour cells, as a proxy for the C2 SCC cancer-stem cell signature, are rare in the papilloma stage and much enriched in the invasive SCC stage

Note: RNAseq in Extended Data Fig

Venn diagram shows the differential expression of genes (DEG) analysis of RNAseq data from TGFβ responding tumour basal cells over their non-responding neighbours and compared between papilloma and SCC. DEG analysis yielded 68 TGFβ responding upregulated genes unique to the papilloma stage, 275 TGFβ responding upregulated genes unique to the SCC stage, and 75 upregulated genes shared by both stages. f, DEG analysis yielded 91 TGFβ responding downregulated genes unique to the papilloma stage, 224 TGFβ responding downregulated genes unique to the SCC stage, and 112 downregulated genes shared by both stages. g, Transcript levels of Lep are below the limits of detection in papillomas and SCCs. And Lepr are also not expressed in normal skin SCs. All statistics were using unpaired two-tailed Student’s t-test: ns, p ≥ 0.05; *, p ≤ 0.05); **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001

Data are presented as mean ± s.e.m

Source data

Data are presented as mean ± s.e.m

Source data

a, LEPR immunofluorescence of human normal skin, head and neck SCC (HNSCC), and lung metastases from human SCC A431 epidermal cells following tail-vein injections in immunocompromised mice

Top row: LEPR labelling alone; bottom row: LEPR, Keratin 14 and DAPI

Data are presented as mean ± s.e.m

a, ATAC sequencing is performed on FACS-purified α6hiβ1hi basal populations of interfollicular epidermis (IFE, Sca1+), bulge hair follicle stem cells (HFSCs, CD34+), and tumour cells (CD44hi) either positive or negative for TGFβ-responsiveness (mCherry)

b, Venn diagram showing marked divergence of ATAC peaks from TGFβ-responding tumour basal cells and their non-responding neighbours between papilloma and SCC stages (n = 2 for each condition, each stage)

d, Quantifications of the Lepr cis-regulatory region boxed in Fig

See also pSMAD2 immunofluorescence quantifications in Extended Data Fig

f, Lepr EGFP reporter and TGFβ mCherry reporter show minimal activity in papillomas but co-localize at the invasive fronts of SCC

Data are presented as mean ± s.e.m

Source data

a, Lepr cis-regulatory region reporter (see Fig. 2g and Extended Data Fig. 5f) was transduced into PDV SCC cells and tested for its sensitivity to TGFβ in vitro

Flow cytometry quantifications show that Lepr reporter-fluorescence is strongly accentuated in the presence of active recombinant TGFβ1 (n = 3, p = 0.0068)

b, Leprnull PDVC57 SCC cells were generated by targeted CRISPR/CAS9 technology and validated by iSeq

Blue denotes sequence comparison region; green sgRNA; red, Lepr frameshift mutation in Clone 2

MiSeq analysis of Lepr targeted Clone 1 (which did not alter LEPR expression), and Clone 3 (which did reduce LEPR expression but not to the extent of Clone 2)

Immunoblot (right) shows complete loss of LEPR protein in this clone, which was selected for further study

For gel source data, see Supplementary Fig

Brackets denote expected sizes of full-length (FL) LEPR and Δsig LEPR, which lacks the LEPR-signalling domain. α-Tubulin is used as loading control. For gel source data, see Supplementary Fig. 2b. All statistics were using unpaired two-tailed Student’s t-test: ns, p ≥ 0.05); *, p ≤ 0.05); **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001

Data are presented as mean ± s.e.m

Source data

b, Tumour growth and angiogenesis are enhanced by systemic recombinant mouse mVEGFA, delivered to the circulation by an osmotic pump distant from PDV SCC tumour sites, which are monitored for about 5W

4g, this finding indicates that elevated levels of plasma leptin on its own is sufficient to enhance tumour growth, independent of possible secondary consequences arising from enhanced angiogenesis that might otherwise bring other hormones or growth factors to the surrounding tumour tissue

d, Immunofluorescence of tissue sections for Lepr reporter and CD31

Quantifications are based on the average distance from the CD31+ vasculature to tumour basal cells with or without reporter signalling

Data are presented as mean ± s.e.m

Source data

pSTAT3 is reduced but not abolished in Leprnull compared to LeprCtrl PDV tumours (right), suggesting that LEPR’s main role in SCC tumour progression is not to activate STAT3

d, Lepr downstream signalling Akt pathway mRNA signature is enriched in C2 cluster of scRNAseq of SCC

f, Leprctrl PDV cells are significantly larger in size compared to Leprnull PDV cells (n = 4 for each condition, p < 0.0001). g, Schematic summarizing our findings. During tumour progression, dynamic crosstalk between HRASG12V oncogenic epithelial SCs and their tumour microenvironment promotes an increase in the production of angiogenesis factors by emerging SCC-CSCs, which in turn fuels angiogenesis, elevating the levels of circulating factors, such as leptin by increasing vasculature density. The perivasculature also raises local immune cells that elevate local TGFβ levels. Enhanced TGFβ-signalling in the CSCs not only promotes an EMT-like invasion6, but also activates Lepr transcription. This triggers a leptin-LEPR signalling cascade, elevating PI3K-AKT-mTORC signalling and fuelling SCC progression. The genes in this cascade are often found mutated in cancers, but as shown here, can be driven by interactions between CSCs and their tumour microenvironment. See also Fig. 5a. All statistics were using unpaired two-tailed Student’s t-test: ns, p ≥ 0.05); *, p ≤ 0.05); **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001

Data are presented as mean ± s.e.m

Source data

Ras drives malignancy through stem cell crosstalk with the microenvironment

Summarized by 365NEWSX ROBOTS

RECENT NEWS

SUBSCRIBE

Get monthly updates and free resources.

CONNECT WITH US

© Copyright 2024 365NEWSX - All RIGHTS RESERVED